USPTO Patents Application 09993647

WORLD INTELLECTUAL PROPERTY ORGANIZATION
International Bureau

PCX

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification 6 ;
C07D 401/04, A61K 31/44

Al

(11) International Publication Number: WO 95/31451

(43) International Publication Date: 23 November 1995 (23.11.95)

(21) International Application Number: PCT/US95/06287

(22) International Filing Date: 16 May 1995 (16.05.95)

(30) Priority Data:

08/242,906

16 May 1994 (16.05.94)

US

(60) Parent Application or Grant

(63) Related by Continuation
US

Filed on

08/242,906 (CIP)
16 May 1994 (16.05.94)

(71) Applicant {for all designated States except US): SMTTHKLINE

BEECHAM CORPORATION [US/US]; Corporate Intellec-
tual Property, UW2220, 709 Swedeland Road, P.O. Box
1539, King of Prussia, PA 19406-0939 (US).

(72) Inventors; and

(75) Inventors/Applicants (for US only): ADAMS, Jerry, Leroy
[US/US]; 611 Forest Road, Wayne, PA 19087 (US).
GALLAGHER, Timothy, Francis [US/US]; 255 Manor
Road, Harleysville, PA 19438 (US). GARIGIPATI, Ravi,
Shanker [IN/US]; 5 Hintlock Lane, Wayne, PA 19087 (US).
THOMSON, Susan, Mary [US/US]; 75 Guilford Circle,
Phoenixville, PA 19460 (US).

(74) Agents: DINNER, Dara, L. et al.; SmithKline Beecham
Corporation, Corporate Intellectual Property, UW2220, 709
Swedeland Road, P.O. Box 1539, King of Prussia, PA
19406-0939 (US).

(81) Designated States: JP, US, European patent (AT, BE, CH, DE,
DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE).

Published

With international search report.

Before the expiration of the time limit for amending the
claims and to be republished in the event of the receipt of
amendments.

(54) Title: NOVEL COMPOUNDS
(57) Abstract

This invention relates to the novel pyrazole compounds of Formula (I), and pharmaceutical compositions comprising a compound
of Formula (I) and a pharmaceutically acceptable diluent or carrier. This invention also relates to a method of inhibiting cytokines by a
compound of Formula (I) and the treatment of cytokine mediated diseases, in mammals, by administration of said compound.

FOR THE PURPOSES OF INFORMATION ONLY

Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
applications under the PCT.

AT

Austria

GB

United Kingdom

MR

Mauritania

AU

Australia

GE

Georgia

MW

Malawi

BB

Barbados

GN

Guinea

NE

Niger

BE

Belgium

GR

Greece

NL

Netherlands

BF

Burkina Faso

HU

Hungary

NO

Norway

BG

Bulgaria

IE

Ireland

NZ

New Zealand

BJ

Benin

IT

Italy

PL

Poland

BR

Brazil

JP

Japan

FT

Portugal

BY

Belarus

KE

Kenya

RO

Romania

CA

Canada

KG

Kyrgystan

RU

Russian Federation

CF

Central African Republic

KP

Democratic People's Republic

SD

Sudan

CG

Congo

of Korea

SE

Sweden

CH

Switzerland

KR

Republic of Korea

SI

Slovenia

CI

Cate d'lvoire

KZ

Kazaldistan

SK

Slovalcia

CM

Cameroon

LI

Liechtenstein

SN

Senegal

CN

Cliina

LK

Sri Lanka

TD

Chad

cs

Czechoslovakia

LU

Luxembourg

TG

Togo

cz

Czech Republic

LV

Latvia

TJ

Tajikistan

DE

Germany

MC

Monaco

TT

Trinidad and Tobago

DK

Denmarlc

MD

Republic of Moldova

UA

Ulaaine

ES

Spain

IVIG

Madagascar

US

United States of America

Fl

Finland

ML

Mali

XJZ

Uzbeldstan

FR

Fiance

MN

Mongolia

VN

Viet Nam

GA Gabon

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NOVEL COMPOUNDS

FIELD OF THE INVENTION

This invention relates to a novel group of pyrazole compounds, processes for the
10 preparation thereof, the use thereof in treating cytokine mediated diseases and
pharmaceutical compositions for use in such therapy.

BACKGROUND OF THE INVENTION:

Interleukin-1 (IL-1) and Tumor Necrosis Factor (TNF) are biological substances

15 produced by a variety of cells, such as monocytes or macrophages. IL-1 has been
demonstrated to mediate a variety of biological activities thought to be important in
immunoregulation and other physiological conditions such as inflammation [See, e.g.,
Dinarello et al.. Rev. Infect. Disease . ^, 51 (1984)]. The myriad of known biological
activities of IL- 1 include the activation of T helper cells, induction of fever, stimulation

20 of prostaglandin or coUagenase production, neutrophil chemotaxis, induction of acute
phase proteins and the suppression of plasma iron levels.

There are many disease states in which excessive or unregulated IL-1 production
is implicated in exacerbating and/or causing the disease. These include rheumatoid
arthritis, osteoarthritis, endotoxemia and/or toxic shock syndrome, other acute or chronic

25 inflammatory disease states such as the inflammatory reaction induced by endotoxin or
inflammatory bowel disease; tuberculosis, atherosclerosis, muscle degeneration,
cachexia, psoriatic arthritis, Reiter's syndrome, rheumatoid arthritis, gout, traumatic
arthritis, rubella arthritis, and acute synovitis. Recent evidence also links IL-1 activity to
diabetes and pancreatic gcells.

3 0 Dinarello, J. Clinical I mmunolof v . 5 (5), 287-297 (1985), reviews tiie biological

activities which have been attributed to IL-1. It should be noted that some of these
effects have been described by others as indirect effects of IL-1.

Excessive or unregulated TNF production has been implicated in mediating or
exacerbating a number of diseases including riieumatoid arthritis, rheumatoid spondyhtis,

3 5 osteoarthritis, gouty arthritis and otiier arthritic conditions; sepsis, septic shock, endotoxic
shock, gram negative sepsis, toxic shock syndrome, adult respiratory distress syndrome,
cerebral malaria, chronic pulmonary inflammatory disease, silicosis, pulmonary
sarcoisosis, bone resorption diseases, reperfusion injury, graft vs. host reaction, allograft
rejections, fever and myalgias due to infection, such as influenza, cachexia secondary to

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infection or malignancy, cachexia, secondary to acquired immune deficiency syndrome
(AIDS), AIDS, ARC (AIDS related complex), keloid formation, scar tissue formation,
Crohn's disease, ulcerative colitis, or pyresis.

AIDS results from the infection of T lymphocytes with Human Immunodeficiency
5 Virus (HIV). At least three types or strains of HIV have been identified, i.e., HIV- 1 ,
HIV-2 and HIV-3. As a consequence of HTV infection, T-cell mediated immunity is
impaired and infected individuals manifest severe opportunistic infections and/or unusual
neoplasms. HIV entry into the T lymphocyte requires T lymphocyte activation. Other
viruses, such as HTV-l, HIV-2 infect T lymphocytes after T Cell activation and such virus

10 protein expression and/or replication is mediated or maintained by such T cell activation.
Once an activated T lymphocyte is infected with HIV, the T lymphocyte must continue to
be maintained in an activated state to permit HIV gene expression and/or HIV replication.
Monokines, specifically TNF, are implicated in activated T-cell mediated HIV protein
expression and/or virus replication by playing a role in maintaining T lymphocyte

15 activation. Therefore, interference with monokine activity such as by inhibition of

monokine production, notably TNF, in an HIV-infected individual aids in limiting the
maintenance of T cell activation, thereby reducing the progression of HTV infectivity to
previously uninfected cells which results in a slowing or elimination of the progression of
immune dysfunction caused by HIV infection. Monocytes, macrophages, and related

20 cells, such as kupffer and glial cells, have also been implicated in maintenance of the HTV
infection. These cells, like T-cells, are targets for viral replication and the level of viral
replication is dependent upon the activation state of the cells. [See Rosenberg etal .. The
Immunopathogenesis of HIV Infection, Advances in Immunology, Vol. 57, (1989)].
Monokines, such as TNF, have been shown to activate HIV replication in monocytes

25 and/or macrophages [See Poli, ct al.. Proc. Nad. Acad. Sci., 87:782-784 (1990)],

therefore, inhibition of monokine production or activity aids in limiting HTV progression
as stated above for T-cells.

TNF has also been implicated in various roles with other viral infections, such as
the cytomegalia virus (CMV), influenza virus, and the herpes virus for similar reasons as

30 those noted.

Interleukin -8 (IL-8) is a chemotactic factor first identified and characterized in
1987. IL-8 is produced by several cell types including mononuclear cells, fibrt)blasts,
endothelial cells, and keratinocytes. Its production fi-om endothelial cells is induced by
IL-1, TNF, or lipopolysachharide (LPS). Human IL-8 has been shown to act on Mouse,
35 Guinea Pig, Rat, and Rabbit Neutrophils. Many different names have been applied to
IL-8, such as neutrophil attractant/activation protein- 1 (NAP-1), monocyte derived

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neutrophil chemotactic factor (MDNCF), neutrophil activating factor (NAF), and T-cell
lymphocyte chemotactic factor.

IL-8 stimulates a number of functions in vitro. It has been shown to have
chemoattractant properties for neutrophils, T-lymphocytes, and basophils. In addition it
induces histamine release from basophils from both normal and atopic individuals as well
as lysozomal enzyme release and respiratory burst from neutrophils. IL-8 has also been
shown to increase the surface expression of Mac-1 (CDl lb/CD18) on neutrophils without
de novo protein synthesis, this may contribute to increased adhesion of the neutrophils to
vascular endothelial cells. Many diseases are characterized by massive neutrophil
infiltration. Conditions associated with an increased in IL-8 production (which is
responsible for chemotaxis of neutrophils into the inflammatory site) would benefit by
compounds which are suppressive of IL-8 production.

IL-1 and TNF affect a wide variety of cells and tissues and these cytokines as well
as other leukocyte derived cytokines are important and critical inflammatory mediators of
a wide variety of disease states and conditions. The inhibition of these cytokines is of
benefit in controlling, reducing and alleviating many of these disease states.

There remains a need for treatment, in this field, for compounds which are
capable of inhibiting cytokines, such as EL-l, IL-6, IL-8 and TNF.

SUMMARY OF THE INVENTION

This invention relates to the novel compounds of Formula d) and pharmaceutical
compositions comprising a compound of Formula (I) and a pharmaceutically acceptable
diluent or carrier.

This invention also relates to a method of inhibiting cytokines and the treatment
of a cytokine mediated disease, in a mammal in need thereof, which comprises
administering to said mammal an effective amount of a compound of Formula (I).

This invention more specifically relates to a method of inhibiting the production
of IL-1 in a mammal in need thereof which comprises administering to said mammal an
effective amount of a compound of Formula (I).

This invention more specifically relates to a method of inhibiting tiie production
of IL-8 in a mammal in need thereof which comprises administering to said mammal an
effective amount of a compound of Formula (I).

This invention more specifically relates to a method of inhibiting the production
of TNF in a mammal in need thereof which comprises administering to said mammal an
effective amount of a compound of Formula (I).

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DETAILED DESCRIPTION OF THE INVENTION

The novel comp>ounds of this invention are represented by the structure having the
formula (I):

N-R3

N

(I)
wherein

one of Ri and R2 is selected from is 4-pyridyl, 4-pyrimidinyl, 4-quinolyl, 4-
isoquinolinyl, 4-quinazolinyl, which is optionally substituted with one or two
substituents each of which is independently selected from C1.4 alkyl, halo, C1-4

1 0 alkoxy, C 1-4 alkylthio, CH2OR8, NH2, mono- or di-Ci -6-alkylamino or N-

heterocyclyl ring which ring has from 5 to 7 members and optionally contains an
additional heteroatom selected from oxygen, sulfur or NR22; and
the other of Ri and R2 is selected from an optionally substituted aryl or optionally
substituted heteroaryl group, provided that both Ri and R2 are not the same

15 heteroaryl group; wherein when one Ri and R2 is an optionally substituted aryl ring,

the ring is substituted by one or two substituents, each of which is independently
selected, and which, for a 4-phenyl, 4-naphth-l-yl or 5-naphth-2-yl substituent, is
halo, nitro, cyano, -C(Z)NR7Ri7, -C(Z)OR23, -(CRl0R20)nCOR36, -SR5, -S(0)R5,
-OR36, halo-substituted C1-4 alkyl, C1.4 alkyl, -ZC(Z)R36, -NRioC(Z)R23, or

20 -(CRioR20)n NR10R2O and which, for other positions of substitution, is halo,

-(CRioR20)n nitro, -(CRioR20)ncyano, -(CRioR20)nC(Z)NRi6R26,
-(CRioR20)nC(ZX)Rl8, -(CRl0R20)n COR25, -(CRioR20)n-S(0)mR8,
-(CRioR20)nOH, -(CRioR20)nOR25. halo-substituted-Ci-4 alkyl, -Ci-4 alkyl,
-(CRi0R20)nNRl0C(Z)R25,-(CRi0R20)nNHS(O)mR6,

25 -(CRioR20)nNHS(0)mNR7Ri7, -(CRioR20)nNR6S(0)mR6,

-(CRioR20)nNR6S(0)m'NR7Rl7; -(CRl0R20)nZC(Z)Ri8 or
-(CRioR20)nNR7Rl7; and when one of Ri and R2 is an optionally substituted
heteroaryl group, the substituent groups include one or two substituents each of which
is independentiy selected from C1-4 alkyl, halo, Ci-4 alkoxy, C1-4 alkylthio,

30 NR10R2O, or an N-heterocyclyl ring which ring has from 5 to 7 members and

optionally contains an additional heteroatom selected from oxygen, sulfur or NR12;
n is 0 or an integer of 1 or 2;
n' is 0 or an integer having a value of 1 to 10;

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n" is an integer having a value of 1 to 10;
m is 0 or an integer of 1 or 2;
m' is an integer of 1 or 2;
m" is an integer having a value of 1 to 10;
5 R3isQ-(Yi)t;

Q is an aryl or heteroaryl group;

t is an integer having a value of 1 to 3;

R4 is hydrogen, Ci-iQalkyl, halo-substituted Ci-io alkyl, C2-IO alkenyl, C2-10 alkynyl,
C3-7 cycloalkyl, Cs-VcycloalkylCi-io alkyl, C5.7 cycloalkenyl, C5-7 cycloalkenyl-

10 Ci-iO alkyl, aryl, arylCi-iQ alkyl, heteroaryl, heteroaryl-Ci-iQ-alkyl, heterocyclyl,

heterocyclylCi- 10 alkyl, -(CRioR20)n' OR12, (CRioR20)nORi3,
(CRioR20)nS(0)mRl8. (CRl0R20)nNHS(O)2Rl8, (CRioR20)nNRi6R26,
(CRi0R20)nNO2, (CRioR20)nCN. (CRioR20)n'S02Rl8,
(CRioR20)nS(0)m'NRi6R26, (CRioR20)nC(Z)Ri3, (CRioR20)nOC(Z)Ri3,

1 5 (CRioR20)nC(Z)ORi3, (CRioR20)nC(Z)NRi6R26. (CRioR20)nC(Z)NRi30R9,

(CRioR20)nNRloC(Z)Ri 1. (CRioR20)nNRioC(Z)NRi6R26,
(CRi0R20)nN(OR6)C(Z)NRi6R26,(CRl0R20)nN(OR6)C(Z)Ri3,

(CRioR20)nC(=NOR6)Rl3.(CRloR20)nNRioC(=NRi9)NRi6R26,
(CRioR20)nOC(Z)NRi6R26, (CRloR20)nNRioC(Z)NRi6R26.
20 (CRioR20)nNRloC(Z)ORio, 5-(Ri8)-l,2,4-oxadizaol-3-yl or 4-(Ri2)-5-(Ri8Ri9)-

4,5-dihydro-l,2,4-oxadiazol-3-yl; wherein die aryl, arylalkyl, heteroaryl, heteroaryl
alkyl, cyclcoalkyl, cycloalkyl alkyl, heterocyclic and heterocyclic alkyl groups may
be optionally substituted;
R6 is Ci-io alkyl, C3-7 cycloalkyl, aryl. arylCi-iQalkyl, heterocyclyl, heterocyclyl
25 Ci-ioalkyl, heterocyclyl-Ci-iQalkyl, heteroaryl or heteroarylCi-iQalkyl;

R5 is hydrogen, Ci-4 alkyl, C2-4 alkenyl, C2-4 alkynyl or NR7R17, excluding the

moieties -SR5 being -SNR7R17 and -SOR5 being -SOH;
RI6 and R26 are independendy hydrogen, or Ci^ alkyl or R16 and R26 together with
the nitrogen to which they are attached form a heterocyclic or heteroaryl ring of 5-7
30 members optionally conatining an additional heteroatom selected from oxygen, sulfur

or NR22;

Yi is independently selected from hydrogen, C1-5 alkyl, halo-substituted C1-5 alkyl,
halogen, or -(CRioR20)nY2;

Y2 is hydrogen, halogen, -ORs, -NO2, -S(0)m'Rl l, -SRs, -S(0)m'0R8, -S(0)niNR8R9,
3 5 -NR8R9, -0(CRioR20)n' NR8R9. -C(0)R8, -CO2R8,

-C02(CRioR20)m"CONR8R9, -ZC(0)R8, -CN, -C(Z)NR8R9, -NRioC(Z)R8,
-C(Z)NR80R9, -NRioC(Z)NR8R9. -NRi0S(O)m'Rll, -N(OR2l)C(Z)NR8R9,

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-N(OR2l)C(Z)R8, -C(=NOR2l)R8, -NRioC(=NRi5)SRii,

-NRioC(=NRi5)NR8R9, -NRioC(=CRi4R24)SRll, -NRioC(=CRi4R24)NR8R9,
-NRi0C(O)C(O)NR8R9, -NRioC(0)C(0)ORio, -C(=NRi3)NR8R9,
-C(=NORi3)NR8R9, -C(=NRi3)ZRii, -OC(Z)NR8R9, -NRioS(0)tnCF3,
5 -NRioC(Z)ORio, 5-(Ri8)-l,2,4-oxadizaol-3-yl or 4-(Ri2)-5-(Ri8Rl9)-4,5-dihydro-

1 ^,4-oxaciiazol-3-yl;

R7 and Ri7 is each independently selected from hydrogen or C 1.4 alkyl or R7 and R17

together with the nitrogen to which they are attached form a heterocyclic ring of 5 to

7 members which ring optionally contains an additional heteroatom selected from
1 0 oxygen, sulfur or NR22;

R8 is hydrogen, Ci-io alkyl, C3-7 cycloalkyl, heterocyclyl, heterocyclylCi-iQalkyl, aryl,

arylCi-ioalkyl, heteroaryl or heteroarylCi-iQalkyl;
R9 is hydrogen, Ci-io alkyl, C2-IO alkenyl, C2-IO alkynyl, C3-7 cycloalkyl, C5.7

cycloalkenyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl or R8 and R9 may together
15 with the nitrogen to which they are attached form a heterocyclic or heteroaryl ring of

5 to 7 members which ring optionally contains an additional heteroatom selected from

oxygen, sulfur or NR 12;
RlO and R20 is each independently selected from hydrogen or C1-4 alkyl;
Rll is Ci-io alkyl, halo-substituted Ci-io alkyl, C2-IO alkenyl, C2-IO alkynyl, C3-7
20 cycloalkyl, C5-7 cycloalkenyl, heterocyclic, heterocyclic Ci-ioalkyl, aryl, aryl

Ci-ioalkyl, heteroaryl or heteroaryl Ci-iQalkyl;
R12 is hydrogen, -C(Z)Ri3, optionally substituted C1-4 alkyl, optionally substituted aryl,

optionally substituted aTyl-Ci-4 alkyl, or S(0)2Rl8J
Rl3 is hydrogen, Ci- 10 alkyl, C3.7 cycloalkyl, heterocyclyl, heterocyclylCi-io alkyl

2 5 aryl, arylC 1 - 1 Qalkyl, heteroaryl or heteroarylC 1 . 1 Qalkyl;

Rl4 and R24 is each independentiy selected from hydrogen, alkyl, nitro or cyano;
Rl5 is hydrogen, cyano, Ci_4 alkyl, C3-7 cycloalkyl or aryl;

RI6 and R26 is each independentiy selected from hydrogen or optionally substituted
Ci-4 alkyl, optionally substimted aryl or optionally substituted aryl-Ci_4 alkyl, or

3 0 together with the nitrogen which they are attached form a heterocyclic ring of 5 to 7

members which ring optionally contains an additional heteroatom selected from
oxygen, sulfur or NR12;
RI8 is Ci-io alkyl, C3-7 cycloalkyl, aryl, arylCi-i Qalkyl, heterocyclyl, heterocyclyl-
Ci-ioalkyl, heteroaryl or heteroarylCi-i Qalkyl;
35 Ri9 is hydrogen, cyano, Ci_4 alkyl, C3-7 cycloalkyl or aryl;

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R21 is hydrogen, a pharmaceutically acceptable cation, Ci-io alkyl, C3-7 cycloalkyl,
aryl, arylCi_4 alkyl, heteroaryl, heteroarylalkyl, heterocyclyl, aroyl, or Ci-io
alkanoyl;

R22 is RlO or C(Z)-Ci-4 alkyl;
5 R23 is Ci-4 alkyl, halo- substituted-C 1.4 alkyl, or C3-7 cycloalkyl;

R25 is Ci-io alkyl, halo-substituted Ci_io alkyl, C2-IO alkenyl, C2-IO alkynyl, C3-7
cycloalkyl, C5.7 cycloalkenyl, aryl, arylCi-io alkyl, heteroaryl, heteroarylCi-iQ
alkyl, heterocyclyl, heterocyclylCi-io alkyl, (CRioR20)nOR8,
(CRioR20)nS(0)inRl8, (CRioR20)nNHS(0)2Rl8, (CRioR20)nNR7Rl7; wherein
10 the aryl, arylalkyl, heteroaryl, heteroaryl alkyl may be optionally substituted;

R36 is hydrogen or R23;

or a pharmaceutically acceptable salt thereof.

Suitable heteroaryl moieties for Ri and R2 are 4-pyridyl, 4-pyrimidinyl,
1 5 4-quinolyl, or 6-isoquinolinyl, all of which may be optionally substituted. Preferably the
heteroaryl group is a 4-pyridyl, 4-pyrimidinyl or 4-quinolyl. More preferred is an
optionally substituted 4-pyrimidinyl or optionally substituted 4-pyridyl moeity, and most
preferred is an optionally substituted 4-pyTimidinyl ring.

20 Suitable substituent groups for the heteroaryl moieties, Ri and R2, include one or

two substituents each of which are independently selected ftx5m C1-4 alkyl, halo,
hydroxy, Ci-4 alkoxy, C1.4 alkylthio, C1-4 alkylsulfinyl, CH2OR8, amino, mono- or di-
Ci-6 alkyl substituted amino, or an N-heterocyclyl ring which ring has from 5 to 7
members and optionally contains an additional heteroatom selected from oxygen, sulfur

25 orNR22.

Prefeired substituents for the heteroaryl moieties Ri is C1.4 alkyl, amino, and
mono-Ci-6alkyl substituted amino. Preferably the C1.4 alkyl is methyl, and for the
mono-Ci-6alkyl substituted amino the alkyl it is of 1-4 carbons in length or shoner, such
as 1 to 2 carbons, preferably methyl. A preferred ring placement of the Ri substiment on
30 the 4-pyridyl derivative is the 2-position, such as 2-methyl-4-pyridyl. A preferred ring
placement on the 4-pyrimidinyl ring is also at the 2-position, such as in 2-methyl-
pyrimidinyl, 2-amino pyrimidinyl or 2-methylaminopyrimidinyl.

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10

15

20

25

For the purposes herein the "core" 4-pyrimidinyl moiety for Ri or R2 is referred

Suitable aryl groups for the other of Ri or R2 include optionally substituted
phenyl, naphth-l-yl or naphth-2-yl. The aryl ring may be optionally substituted by one or
two substituents, each of which is independently selected, and which, for a 4-phenyl, 4-
naphth-l-yl or 5-naphth-2-yl substitucnt, is halo, nitro, cyano, -C(Z)NR7Ri7,
-C(Z)OR23. -(CRl0R20)nCOR36, -SR5, -S(0)R5, -OR36, halo-substituted Ci-4 alkyl,
Cl-4 alkyl, -ZC(Z)R36, -NRioC(Z)R23. or -(CRioR20)n NR10R2O and which, for other
positions of substitution, is halo, nitro, cyano, -C(Z)NRi6R26. C(Z)ORi8, -(CRl0R20)n
COR25. -S(0)mR8. OH, -OR25, halo-substituted-Ci-4 alkyl, -C1.4 alkyl,
-(CRi0R20)nNRl0C(Z)R25, -NHS(0)mR6, -NHS(0)mNR7Rl7, -NR6S(0)mR6>
-NR6S(0)m'NR7Rl7 wherein m' is 1 or 2, -ZC(Z)Ri8 or -(CRioR20)n NR7R17.
Preferably the aryl ring is a phenyl which is optionally substituted.

Preferred substitutions for the Ri or R2 group when it is a 4-phenyl, 4-naphth-
1-yl or 5-naphth-2-yl moiety are one or two substituents each independendy selected
from halogen, -SR5, -SOR5, -OR36, or -(CRioR20)nNRioR20, and for other positions
of substitution on these rings preferred substitution is halogen, -S(0)mR25. -OR25,
-(CRi0R20)nNR7Rl7, -(CRloR20)nNRioC(Z)R25 and -NR6S(0)m*R6. More
preferred substituents for the 4-position in phenyl and naphth-l-yl and on the 5-position
in naphth-2-yl include halogen, especially fluoro and chloro, more especially fluoro, and
-SR5 and -S(0)R5 wherein R5 is preferably a Ci-2 alkyl, more preferably methyl; of
which fluoro is especially preferred.

Preferred substituents for the 3-position in phenyl and naphth-l-yl include:
halogen, especially chloro; -OR25, especially C1-4 alkoxy; amino; -NRioC(Z)R25,
especially -NHCO(Cl-ioalkyl); and -NRioS(0)m'R6, especially -NHSO2(Ci-i0alkyl).
Preferably, the aryl group is an unsubstituted or substituted phenyl moiety. More
preferably, it is phenyl or phenyl substituted at the 4-position with fluoro and/or
substituted at the 3-position with fluoro, chloro, Ci-4 alkoxy, methanesulfonamido or
acetamido.

to as the formula:

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Suitably, R3 is Q-(Yi)t wherein Q is an aryl or heteroaiyl group; and t is an
integer having a value of 1 to 3. Suitably Yi is independently selected from hydrogen,
Ci-5 alkyl, halo-substituted C1-5 alkyl, halogen, or -(CRioR20)nY2.

Suitably Y2 is hydrogen, halogen, -ORg, -NO2, -S(0)m'Rll, -SRg, -S(0)m'0R8,
5 -S(0)mNR8R9, -NR8R9. -0(CR 10R20)nNR8R9> -C(0)R8, -CO2R8,

-CO2(CRl0R20)ni" CONR8R9. -ZC(0)R8, -CN, -C(Z)NR8R9, -NRioC(Z)R8,
-C(Z)NR80R9, -NRioC(Z)NR8R9, -NRioS(0)mRll, -N(OR2l)C(Z)NR8R9,
-N(OR2l)C(Z)R8, -C(=NOR2l)R8. -NRioC(=NRi5)SRii, -NRioC(=NRi5)NR8R9,
-NRioC(=CRi4R24)SRll, -NRioC(=CRi4R24)NR8R9. -NRioC(0)C(0)NR8R9.
10 -NRioC(0)C(0)ORio, -C(=NRi3)NR8R9, -C(=NORi3)NR8R9, -C(=NRi3)ZRi 1,

-OC(Z)NR8R9, -NRioS(0)niCF3, -NRioC(Z)ORio, 5-(Ri8)-l,2,4-oxadizaol-3-yl or 4-
(R 1 2)-5-(Rl gR 1 9)-4,5-dihydiD- 1 ,2,4-oxadiazol-3-yl.

Preferably Q is phenyl which is optionally substitued. Preferable substituents
include -(CRioR20)nY2 with Y2 as -S(0)m'Rll. -SR8, halogen or -CO2R8; n is
1 5 preferably 0 or 1 .

Preferably t is 1 or 2. More preferably, when R3 is monosubstituted phenyl
(t=l), the substituent is located at the 4-position.

Suitably R4 is hydrogen, Ci-ioalkyl, halo-substituted Ci-io alkyl, C2-IO
20 alkenyl, C2-IO alkynyl, C3-7 cycloalkyl, C3-7cycloalkylCi.io alkyl, C5.7 cycloalkenyl,
C5-7cycloalkenyl-Ci-iO alkyl, aryl, arylCi-io alkyl, hcteroaryl, heteroaryl-Ci-iQ-alkyl,
heterocyclyl, heterocyclylCi-io alkyl, -(CRioR20)n' OR12, (CRioR20)nORi3,
(CRioR20)nS(0)mRl8. (CRl0R20)nNHS(O)2Rl8. (CRl0R20)nNRi6R26.
(CRioR20)nN02, (CRioR20)nCN, (CRioR20)n'S02Rl8,
25 (CRi0R20)nS(O)ni'NRi6R26. (CRl0R20)nC(Z)Ri3, (CRioR20)nOC(Z)Ri3,

(CRioR20)nC(Z)ORi3. (CRioR20)nC(Z)NRi6R26, (CRioR20)nC(Z)NRi30R9,
(CRioR20)nNRloC(Z)Ri 1, (CRioR20)nNRioC(Z)NRi6R26.
(CRi0R20)nN(OR6)C(Z)NRi6R26,(CRl0R20)nN(OR6)C(Z)Ri3,
(CRioR20)nC(=NOR6)Rl3,(CRloR20)nNRioC(=NRi9)NRi6R26.
30 (CRioR20)nOC(Z)NRi6R26, (CRl0R20)nNRi0C(Z)NRi6R26,

(CRi0R20)nNRl0C(ZX)Rl0, 5-(Ri8)-l,2,4-oxadizaol-yl or 4-(Ri2)-5-Ri8Ri9)-,5-
dihydro-l,2,4-oxadiazol-3-yl; wherein the aryl, arylalkyl, heteroaryl, heteroaryl alkyl,
cyclcoalkyl, cycloalkyl alkyl, heterocyclic and heterocyclic alkyl groups may be
optionally substituted;

35

More suitably R4 is hydrogen, NRi6R26> NRioC(Z)Rii, NRioC(Z)NRi6R26,
NRioC(=NRi9)NR6R26. or NRioC(Z)ORio. Preferably, R4 is hydrogen or NRi6R26-

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Preferably the Ri6 and R26 groups are independently hydrogen, or Ci_4 alkyl or Ri6
and R26 together with the nitrogen to which they are attached form a heterocyclic or
heteroaryl ring of 5-7 members optionally containing an additional heteroatom selected
from oxygen, sulfur or NR22- The heterocyclic and heteroaryl ring may also be
5 additionally substituted.

In all instances herein where there is an alkenyl or alkynyl moiety as a substituent
group, the unsaturated linkage, i.e., the vinylene or acetylene linkage is preferably not
directly attached to the nitrogen, oxygen or sulfur moieties, for instance in C(Z)NR80R9,
10 NR ioC(Z)NR8R9, or OR25.

As used herein, "optionally substituted" unless specifically defined shall mean
such groups as halogen, such as fluorine, chlorine, bromine or iodine; hydroxy; hydroxy
substituted Ci_ioalkyl; Ci-io alkoxy, such as methoxy or ethoxy; S(0)m Ci-io alkyl,

15 wherein m is 0, 1 or 2, such as methyl thio, methylsulfinyl or methyl sulfonyl; amino,
mono & di-substituted amino, such as in the NR7R17 group; or where the R7R17 may
together with the nitrogen to which they are attached cyclize to form a 5 to 7 membered
ring which optionally includes an additional heteroatom selected from O/N/S; Ci-io
alkyl, cycloalkyl, or cycloalkyl alkyl group, such as methyl, ethyl, propyl, isopropyl,

20 t-butyl, etc. or cyclopropyl methyl; halosubstituted Ci-io alkyl, such CF3; an optionally
substituted aryl, such as phenyl, or an optionally substituted arylalkyl, such as benzyl or
phenethyl, wherein these aryl moieties may also be substituted one to two times by
halogen; hydroxy; hydroxy substituted alkyl; Ci-iQ alkoxy; S(0)m alkyl; amino, mono
and di-substituted amino, such as in the NR7R17 group; Ci-io alkyl, or halosubstituted

25 alkyl, such as CF3.

In a preferred subgenus of compounds of formula (I), Ri is 4-pyridyl, 2-alkyl-4-
pyridyl, 2-NRioR20-4-pyridyl, 4-pyrimidinyl, 2-alkyl-4-pyrimidinyl, 2-NRioR20-4-
pyrimidinyl, wherein one of R10R2O is hydrogen, or 4-quinolyl; more preferably Ri is
30 2-amino-4-pyTimidinyl or 2 methylamino-4-pyrimidinyl; R2 is an optionally substituted
phenyl group. More preferably R2 is phenyl or phenyl substituted by fluoro, chloro, C1-4
alkoxy, S(0)mCi-4 alkyl, methanesulfonamido or acetamido; R3 is phenyl or optionally
substituted phenyl and R4 is hydrogen or NRi6R26-

35 Suitable pharmaceutically acceptable salts are well known to those skilled in the

art and include basic salts of inorganic and organic acids, such as hydrochloric acid,
hydrobromic acid, sulphuric acid, phosphoric acid, methane sulphonic acid, ethane

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sulphonic acid, acetic acid, malic acid, tartaric acid, citric acid, lactic acid, oxalic acid,
succinic acid, fumaric acid, maleic acid, benzoic acid, salicylic acid, phenylacetic acid
and mandelic acid. In addition, phamiaceutically acceptable salts of compounds of
formula (I) may also be formed with a pharmaceutically acceptable cation, for instance, if
5 a substituent Yj in R3 comprises a carboxy group. Suitable pharmaceutically acceptable
cations are well known to those skilled in the art and include alkaline, alkaline earth,
ammonium and quaternary ammonium cations.

The following terms, as used herein, refer to:
10 - "halo"'- all halogens, that is chloro, fluoro, bromo and iodo;

- "Ci.jo alkyl" or "alkyl" - both straight and branched chain radicals of 1 to 10

carbon atoms, unless the chain length is otherwise limited, including, but not limited to,
methyl, ethyl, w-propyl, iso-propyl, /i-butyl, sec-huty\, iso-butyl, tert-hutyl, and the like;

- The term "cycloalkyl" is used herein to mean cyclic radicals, preferably of 3 to
15 7 carbons, including but not limited to cyclopropyl, cyclopentyl, cyclohexyl, and the like.

- The term "alkenyl" is used herein at all occurrences to mean straight or
branched chain radical of 2-10 carbon atoms, unless the chain length is limited thereto,
including, but not limited to ethenyl, 1-propenyl, 2-propenyl, 2-methyl-l-propenyl,
1-butenyl, 2-butenyl and the like.

20 - "aryl" - phenyl and naphthyl;

- "heteroaryl" (on its own or in any combination, such as "heteroaryloxy") - a
5-10 membered aromatic ring system in which one or more rings contain one or more
heteroatoms selected from the group consisting of N, O or S, such as, but not limited
to pyrrole, thiophene, quinoline, isoquinoline, pyridine, pyrimidine, oxazole, thiazole,

2 5 thiadiazole, triazole, imidazole, or benzimidazole;

- "heterocyclic" (on its own or in any combination, such as "heterocyclylalkyl") -
a saturated or wholly or partially unsaturated 4-10 membered ring system in which one or
more rings contain one or more heteroatoms selected firom the group consisting of N, O,
or S; such as, but not limited to, pyrrolidine, piperidine, piperazine, morpholine,

30 imidazolidine or pyrazolidine;

- The term "aralkyl" or "heteroarylalkyl" or "heterocyclicalkyl" is used herein to
mean C1-4 alkyl as defined above unless otherwise indicated

- "sulfinyl" ' the oxide S(0) of the corresponding sulfide while the term "thio"
refers to the sulfide.

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The compounds of the present invention may contain one or more asymmetric
carbon atoms and may exist in racemic and optically active forms. All of these
compounds are included within the scope of the present invention.

5 Compounds of Formula (I) are pyrrazole derivatives which may be readily

prepared using procedures well known to those of skill in the art and may be prepared by
analogous methods to those indicated herein below.

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5

While the illustration in Scheme I is for the preparation of a particular compound
of Formula (I) (i.e.. Scheme I, Ri = pyridyl, R2=4-fluorophenyl, R3 =
methylsulfinylphenyl and R4 is amino or hydrogen), generalization of the synthesis to

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groups claimed as Ri, R2, R3 and R4 herein can be achieved by starting with the
appropriate propanenitrile, preparation of which are disclosed in EP 0 531 901 A2 whose
disclosure is incorporated by reference herein. Treatment of the propanenitrile with the
appropriate arylhydrazine affords 5-aminopyrazole, 3 -Scheme 1 as outlined by Smith,
5 P.A.S. et al., /. Org. Chem., 1970 , 35(7), 2215. Pyrazole 3-Scheme 1 can be converted
to the corresponding sulphonamide, amide, urea, guanidine or urethane by using
techniques well known to those of sldll in the art by the appropriate acylating agents,
such as sulfonyl chlorides, acid chlorides, isocyanates, dicyanamides and chloroformates,
resp>ectively. 5-Aminopyrazole, 3 -Scheme 1 can also be converted to the
10 5-diazopyrazole, 4-Scheme 1 by treatment with NaN02 and aqueous HCl. Following the

procedure of Hodgson, H.H. et al., J. Chem. So. London, 1944, 8, treatment of 4-Scheme 1
with Cu20 and HOAC/H2SO4) affords pyrazole 5-Scheme I.

Alternatively any acylsubstituted malonate ester can be reacted with a substituted
15 hydrazine derivative to afford a 3-hydroxypyrazole such as 1-Scheme 2. A 3-

hydroxypyrazole can be either deoxygenated as shown in scheme 11, or can also be
converted to afford a suitable derivative. The ester functionality can be transformed into
a methyl ketone such as 2-Scheme 2 which can be reacted with a variety of reagents like
Bredrick's reagent or DMF dimethylacetal, to yield an enamine such as 3-Scheme 2.
20 Enamines Qikc 3-Scheme 2) react efficiently with guanidine derivatives (or substituted
guanidine derivatives ) to afford the corresponding 2-aminopyrimidine 4-Scheme 2.

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Scheme II

In addition to reaction with a thiol as illustrated in Scheme n, the triflate of
5 compound l-Scheme n can be reacted with both carbon and nitrogen nucleophiles to
obtain compounds having R4 not equal to hydrogen. For example, the triflate of 1-
Scheme 11 can be displaced with either azide or cyanide to afford comjx)unds which are
themselves intermediates for the synthesis of compounds having respectively either a
nitrogen (amines, amides, ureas) or carbon (ketones, esters, amides, alcohols) attachment
10 to the pyrazole ring. Furthermore the triflate or the halo (chloro, bromo, or iodo)

derivative, which may be prepared either from 1-Scheme 11 directly or firom nucleophilic
displacement of the triflate by the halo group, can be reacted under a variety of transition

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metal (Pd, Ni, etc.) catalyzed cross coupling conditions with a suitable organometallic
based carbon nucleophiles (arylstannanes, boronic acids, organozincs) to afford R4 as an
aryl, heteroaryl, olefin, acetylene, ester, or alkyl substituent.

5 Alternatively, reaction of 3 -Scheme II with S-methyl thiourea produces a 2-

thiomethyl substituted pyrimidine (4-Scheme II with S-methyl in place of NH2). This
intermediate may be reacted under basic conditions with amine nucleophiles to produce
analogs of 4-Scheine n (RHN in place of NH2 wherein R is a suitable substituent group,
such as R13, S(0)2R. etc.) or preferably oxidized to the sulfoxide or sulfone which then
10 undergo more facile displacement with amine nucleophiles to afford the analogs of 4-
Scheme II.

Suitable protecting groups for use in the present invention, are well known in the
art and described in many references, for instance. Protecting Groups in Organic
1 5 Synthesis, Greene T W, Wiley-Interscience, New York, 1981.

Pharmaceutically acid addition salts of compounds of formula (I) may be obtained
in known manner, for example by treatment thereof with an appropriate amount of acid in
the presence of a suitable solvent.

20

The invention will now be described by reference to the following examples
which are merely illustrative and are not to be construed as a limitation of the scope of
the present invention.

25 Synthetic Examples

EXAMPLE 1

3-(4-Fluorophenyl)-l-(4-methylsulfinyIphenyl)-4-(4-pyridyI)-5H-pyrazole

30 (a) 4-(Methylthio)phenylhydrazine hydrochloride - The title compound is prepared
following the pnxedure of Demers, J.P. et al., Tet. Lett., 1987, 28 (42), 4933.

(b) 3.(4-FluorophenyI)-3-oxo-2-(pyridin-4-yl)propanenitriIe - The tide compound is
prepared following the procedure of Oku, T. et al, EP 0 531 901 A2.

(c) 5-Amino-3-(4-nuorophenyl)-l-(methyIthiophenyl)-4-(4-pyridyl)pyrazoIe - The

3 5 title compound is prepared following the procedure of Smith, P.A.S. et al., J. Org. Chem.,
1970, 35 (7), 2215 except using 4-(methylthio)phenylhydrazine hydrochloide and 3-(4-
fluorophenyl)-3-oxo-2-(pyridin-4-yl)propanenitriIe.

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(d) 5-Diazo-3-(4-fluorophenyl)-l-(niethylthiophenyl)-4-(4-pyridyl)pyrazoIe - 5-

Amino-3-(4-fluorophenyl)-l-(methylthiophenyl)-4-(4-pyridyl)pyrazole is dissolved in a
suitable volume of water containing 2.5-3 equivalents of HCl. The solution is cooled in
ice and maintained at 0-5° C while an aqueous soluton of NaN02 is added porionwise.
5 The NaN02 is added until, after 3-4 min, the solution gives a positive result with a moist
potassium iodide-starch paper test. The title compound is used without further
purification.

(e) 3-(4-FluorophenyI)-l-(inethyIthiophenyI)-4-(4-pyridyl)-5H-pyrazole - The title
compound is prepared following the procedure of Hodgson, H.H. et al., J. Chem. Soc.

10 London, 1944, 8, except using 5-diazo-3-(4-fluorophenyl)-l-(methylthiophenyl)-4-(4-
pyridyl)pyrazole.

(f) 3-(4-FIuorophenyI)-l-(4-methylsulfinyIphenyI)-4-(4-pyridyI)-5H-pyrazole - A

solution of 3-(4-fluorophenyl)-l-(methylthiophenyl)-4-(4-pyridyl)-5H-pyrazole in glacial
acetic acid is added to an aqueous solution of K2S2O8. The solution is stirred at rt for 18
15 h. The mixture is then poured into H2O and the pH is adjusted to neutral with cone.
NH4OH. The solid which is formed is collected to afford the title compound.

Examnle 2

20 4-f2-Amino-4-nvriniidinvn-3-f4-ffliioronhenvlVl-nhenvlnvra7nle

a) Diethvl-M-fluorobenzovnmalonate

The title compound was prepared following the procedure of Lawesson, et al,
Acta Chem. Scand., 1959, 13 (8), 1717.

b) 5-Hvdroxv-4-carboethoxv-3-f4-fluoronhenvn.1-nhpnvlnvraTnlP

25 Phenyl hydrazine (1 1.2 mL) was dissolved in 70% acetic acid/water (37 mL) and

added dropwise to a solution of the compound of example 1(a) (6.38 g, 22.6 mmol)
dissolved in glacial acetic acid (37 mL). The resulting mixture was allowed to stand at
room temperature for 3 days before diluting with water and filtering. The resulting
brown crystals were washed with water to afford the title compound (6.41 g, 87% yield).

30 1h NMR (400 MHz, CDCI3): d 7.89 (d, 2H), 7.80 (q, 2H), 7.49 (t, 2H), 7.36 (t, IH),
7.12 (t, 2H), 4.36 (q, 2H), 1.33 (t, 3H).

c) 4-Ethoxvcarbonvl-3-(4-fluoroDhenvn-l-DhenvlDvra7ole

The compound of example 1(b) (.30 g, .92 mmol) and 2,6-lutidine, (.14 mL, 1.20
mmol) in CH2CI2 were cooled to -30 °C. Trifluoromethane sulfonic anhydride (.17 mL,
35 1.01 mmol) was added and the mixture was allowed to stand at 0°C for 24 h. The
mixture was filtered and concentrated and the resulting oil was triturated with 5%
EtOAc/Hexane until the remaining residue solidified. The organic washings were

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combined and concentrated. The resulting oil was combined with 1-propanethiol (2 eq.)
and K2CO3 (2 eq.) in DMF and stirred at room temperature 24 h. The mixture was
diluted with water and extracted with EtOAc. The organic phase was dried (Na2S04)
and concentrated. The resulting residue was purified by flash chromatography (silica gel,
5 0% - 2% acetone/hexane). The isolated product was dissolved in ethanol and combined
with activated Raney Nickel. The mixture was stirred 1 h. and was filtered through celite
rinsing well with ethanol. Evaporation of the solvent afforded the title compound as a
white solid (40% yield). 1h NMR (400 MHz, CDCI3): d 8.50 (s, IH), 7.90 (q, 2H), 7.78
(d, 2H), 7.51 (t, 2H), 7.39 (t, IH), 4.32 (q, 2H), 1.35 (t, 3H).

10 d) N.O-dimethvl-3-r4-fluoroDhenvl)- l-Dhenvlnvra?:ole-4.hvdroxamate

A suspension of N,0-dimethylhydroxylamine hydrochloride (.39 g) in toluene (2
mL) was cooled to 0°C. Trimethylaluminum (2 mL, 2 M solution on toluene) was added
slowly and the mixture was warmed to room temperature and stirred 1 h. This reagent
(1.3 mL, 1.70 mmol) was added to the compound of example 1(c) (.2 g, .68 mmol)

15 dissolved in toluene (4 mL). After stirring at SO^C for 2 h. the reaction was quenched by
the addition of 3 N HCl and the product was extracted with EtOAc. The organic phase
was dried (Na2S04) and concentrated. The residue was suspended in 5% EtOAc/hexane
and the resulting solid was filtered to afford the title compound as a white solid (.15 g,
67% yield). Ir NMR (400 MHz, CDCI3): d 8.35 (s, IH), 7.78 (m, 4H), 7.50 (t, 2H),

20 7.34 (t, IH), 7.11 (t, 2H), 3.60 (s, 3H), 3.31 (s, 3H).

e) 4-acetvl-3-(4-fliiornnhenvn-1-nh envlnvra3:olp

The compound of example 1(d) (.11 g, .34 mmol) in dry THE (7 mL) was cooled
to -78° C. Methylmagnesium bromide (1.36 mmol, 3.0 M sin. in diethyl ether)was added
and the solution was warmed to room temperature. The reaction was quenched with
25 saturated, aqueous NH4CI . The product was extracted with EtOAc. The organic phase
was dried (Na2S04) and concentrated to yield the tide compound (.09 g, 97% yield). ^H
NMR (400 MHz, CDCI3): d 8.74 (s, IH), 7.70 (m, 4H), 7.51 (t. 2H), 7.40 (t, IH), 7.15 (t,
2H), 2.47 (s, 3H).

f) 4rf3-N.N-dimethvn-nrOD-2-ene-1-onel-3-f4.f1iinrnn henvn.l-nhpnvlnvrazole

30 Added to the compound of example 1(e) (.09 g, .33 mmol) in DMF (5 mL) was

fen-butylbis(dimethylamino)methane (.20 mL, .99 mmol). The mixture was stirred at
1 10° C for 4 h. and then concentrated. The resulting residue was purified by flashed
chromatography eluting with 2% MeOH/CH2Cl2 to afford the tide compound (.1 1 g,
99% yield). ^H NMR (400 MHz, CDCI3): d 8.35 (s, IH), 7.78 (m, 4H), 7.65 (d, IH),

3 5 7.45 (t, 2H), 7.29 (t, IH), 7.08 (t, 2H), 5.20 (d, IH), 3.03 (s, 3H), 2.66 (s, 3H).

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g) 4-f2-aniino-4-nvrimidinvn-3-(4-fluoroDhenvlVl-DhenvlDvrazole

Sodium (.0075 g, .33 mmol) was dissolved in ethanol (1 mL). Guanidine
hydrochloride (.03 g, .33 mmol) was added and the mixture was stirred 10 min. The
compoimd of example 1(f) (-1 1 g. .33 mmol) was dissolved in ethanol (5 mL) and added
5 to the mixture and refluxed for 18 h. The resulting mixture was cooled and concentrated.
The residue was triturated with CH2CI2 and filtered. The organic extract was
concentrated. The product was crystallized from ethanol to afford the title comjxjund
(.05 g, 52% yield), mp 170 - 171«C. NMR (400 MHz, CDCI3): d 8.51 (s, IH), 8.16
(d, IH), 7.80 (d, 2H), 7.62 (q, 2H), 7.50 (t, 2H), 7.36 (t, IH), 7.14 (t, IH), 6.49 (d, IH),
10 5.01 (s, 2H).

METHODS OF TREATMENT

The compounds of Formula (I) or a pharmaceutically acceptable salt thereof can
be used in the manufacture of a medicament for the prophylactic or therapeutic treatment
15 of any disease state in a human, or other mammal, which is exacerbated or caused by
excessive or unregulated cytokine production by such mammal's cell, such as, but not
limited to monocytes and/or macrophages.

Compounds of formula (I) are capable of inhibiting proinflammatory cytokines,
such as IL-1, IL-6, IL-8 and TNF and are therefore of use in therapy. IL-1, IL-8 and TNF
20 affect a wide variety of cells and tissues and these cytokines, as well as other leukocyte-
derived cytokines, arc important and critical inflammatory mediators of a wide variety of
disease states and conditions. The inhibition of these pro-inflammatory cytokines is of
benefit in controlling, reducing and alleviating many of these disease states.

Accordingly, the present invention provides a method of treating a cytokine-
25 mediated disease which comprises administering an effective cytokine-interfering amount
of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

In particular, comp>ounds of formula (I) or a pharmaceutically acceptable salt
thereof are of use in the prophylaxis or therapy of any disease state in a human, or other
mammal, which is exacerbated by or caused by excessive or unregulated IL-1, IL-8 or
30 TNF production by such mammal's cell, such as, but not limited to, monocytes and/or
macrophages.

Accordingly, in another aspect, this invention relates to a method of inhibiting the
production of IL-1 in a mammal in need thereof which comprises administering to said
mammal an effective amount of a compound of formula (I) or a pharmaceutically
3 5 acceptable salt thereof.

There are many disease states in which excessive or unregulated BL-l production
is implicated in exacerbating zmd/or causing the disease. These include rheumatoid

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arthriris, osteoarthritis, endotoxemia and/or toxic shock syndrome, other acute or chronic
inflammatory disease states such as the inflammatory reaction induced by endotoxin or
inflammatory bowel disease, tuberculosis, atherosclerosis, muscle degeneration, multiple
sclerosis, cachexia, bone resorption, psoriatic arthritis, Reiter's syndrome, rheumatoid
5 arthritis, gout, traumatic arthritis, rubella arthritis and acute synovitis. Recent evidence
also links IL-1 activity to diabetes, pancreatic gcells and Alzheimer's disease.

In a further aspect, this invention relates to a method of inhibiting the production
of TNF in a mammal in need thereof which comprises administering to said mammal an
effective amount of a compound of formula (I) or a pharmaceutically acceptable salt
10 thereof.

Excessive or unregulated TNF production has been implicated in mediating or
exacerbating a number of diseases including rheumatoid arthritis, rheumatoid spondylitis,
osteoarthritis, gouty arthritis and other arthritic conditions, sepsis, septic shock, endotoxic
shock, gram negative sepsis, toxic shock syndrome, adult respiratory distress syndrome,

15 cerebral malaria, chronic pulmonary inflammatory disease, silicosis, pulmonary

sarcoisosis, bone resorption diseases, such as osteoporosis, reperfusion injury, graft vs.
host reaction, allograft rejections, fever and myalgias due to infection, such as influenza,
cachexia secondary to infection or malignancy, cachexia secondary to acquired immune
deficiency syndrome (AIDS), AIDS, ARC (AIDS related complex), keloid fonnation,

20 scar tissue formation, Crohn's disease, ulcerative colitis and pyresis.

Compounds of formula (I) are also useful in the treatment of viral infections,
where such viruses are sensitive to uprcgulation by TNF or will elicit TNF production in
vivo. The viruses contemplated for treatment herein are those that produce TNF as a
result of infection, or those which are sensitive to inhibition, such as by decreased

25 replication, directiy or indirecdy, by the TNF inhibiting-compounds of formula (1). Such
viruses include, but are not limited to HIV-1, HIV-2 and HIV-3, Cytomegalovirus
(CMV), Influenza, adenovirus and the Herpes group of viruses, such as but not limited to.
Herpes Zoster and Herpes Simplex. Accordingly, in a further aspect, this invention
relates to a method of treating a mammal, preferably a human, afflicted with a human

30 immunodeficiency virus (HTV) which comprises administering to such mammal an
effective TNF inhibiting amount of a compound of formula (I) or a pharmaceutically
acceptable salt thereof.

Compounds of formula (I) may also be used in association with the veterinary
3 5 treatment of mammals, other than in humans, in need of inhibition of TNF production.
TNF mediated diseases for treatment, therapeutically or prophylactically, in animals
include disease states such as those noted above, but in particular yiral infections.

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Examples of such viruses include, but are not limited to, the lentivirus infections such as
equine infectious anaemia virus, caprine arthritis virus, visna virus, or the maedi virus, or
the retroviruses, such as feline immunodeficiency virus (FTV), bovine immunodeficiency
virus, or canine immunodeficiency virus.
5 The compounds of formula (I) may also be used topically in the treatment or

prophylaxis of topical disease states mediated by or exacerbated by excessive cytokine
production, such as by IL-1 or TNF respectively, such as inflamed joints, eczema,
psoriasis and other inflammatory skin conditions such as sunburn; inflammatory eye
conditions including conjunctivitis; pyresis, pain and other conditions associated with
10 inflammation.

Another aspect of the present invention relates to a method of inhibiting the
production of IL-8 (Interleukin-8, NAP) in a mammal in need thereof which comprises
administering to said mammal an effective amount of a compound of formula (I) or a

1 5 pharmaceutically acceptable salt thereof.

There are many disease states in which excessive or unregulated IL-8 production
is implicated in exacerbating and/or causing the disease. These diseases are characterized
by massive neutrophil infiltration such as, psoriasis, inflammatory bowel disease, asthma,
cardiac and renal reperfusion injury, adult respiratory distress syndrome, thrombosis and

20 glomerulonephritis. All of these diseases are associated with increased IL-8 production
which is responsible for the chemotaxis of neutrophils into the inflammatory site. In
contrast to other inflammatory cytokines (IL-1, TNF, and IL-6), IL-8 has the unique
property of promoting neutrophil chemotaxis and activation. Therefore, the inhibition of
IL-8 production would lead to a direct reduction in the neutrophil infiltration.

2 5 The compounds of formula (I) are administered in an amount sufficient to inhibit

cytokine, in particular IL-1, IL-8 or TNF, production such that it is regulated down to
normal levels, or in some case to subnormal levels, so as to ameliorate or prevent the
disease state. Abnormal levels of IL-1, IL-8 or TNF, for instance in the context of the
present invention, constitute: (i) levels of ft-ee (not cell bound) IL-1, IL-8 or TNF greater
30 than or equal to 1 picogram per ml; (ii) any cell associated IL-1 , IL-8 or TNF; or (iii) the
presence of IL-1, IL-8 or TNF mRNA above basal levels in cells or tissues in which IL-1,
IL-8 or TNF, respectively, is produced.

The discovery that the compounds of formula (I) are inhibitors of cytokines,
specifically IL-1, IL-8 and TNF is based upon the effects of the compounds of formula

3 5 (I) on the production of the IL-1 , IL-8 and TNF in in vitro assays which are described

herein.

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As used herein, the term "inhibiting the production of IL-1 (IL-8 or TNF)" refers

to:

a) a decrease of excessive in vivo levels of the cytokine (EL-l, IL-8 or TNF) in a
human to normal or sub-normal levels by inhibition of the in vivo release of the cytokine

5 by all cells, including but not limited to monocytes or macrophages;

b) a down regulation, at the genomic level, of excessive in vivo levels of the
cytokine (IL-1, IL-8 or TNF) in a human to normal or sub-normal levels;

c) a down regulation, by inhibition of the direct synthesis of the cytokine (IL-1,
IL-8 or TNF) as a postranslational event; or

10 d) a down regulation, at the translational level, of excessive in vivo levels of the

cytokine (IL-1, IL-8 or TNF) in a human to normal or sub- normal levels.

As used herein, the term "TNF mediated disease or disease state" refers to any and
all disease states in which TNF plays a role, either by production of TNF itself, or by
TNF causing another monokine to be released, such as but not limited to IL-1, IL-6 or IL-

15 8. A disease state in which, for instance, IL-1 is a major component, and whose

production or action, is exacerbated or secreted in response to TNF, would therefore be
considered a disease stated mediated by TNF.

As used herein, the term "cytokine" refers to any secreted polypeptide that affects
the functions of cells and is a molecule which modulates interactions between cells in the

20 immune, inflammatory or hematopoietic response. A cytokine includes, but is not limited
to, monokines and lymphokines, regardless of which cells produce them. For instance, a
monokine is generally referred to as being produced and secreted by a mononuclear cell,
such as a macrophage and/or monocyte. Many other cells however also produce
monokines, such as natural killer cells, fibroblasts, basophils, neutrophils, endothelial

25 cells, brain astrocytes, bone marrow stromal cells, epideral keratinocytes and B-

lymphocytes. Lymphokines are generally referred to as being produced by lymphocyte
cells. Examples of cytokines include, but are not limited to, Interleukin- 1 (IL-1),
Interleukin-6 (IL-6), Interleukin-8 (IL-8), Tumor Necrosis Factor-alpha (TNF-a) and
Tumor Necrosis Factor beta (TNF- B) .

30 As used herein, the term "cytokine interfering" or "cytokine suppressive amount"

refers to an effective amount of a compound of formula (1) which will cause a decrease in
the in vivo levels of the cytokine to normal or sub-normal levels, when given to a patient
for the prophylaxis or treatment of a disease state which is exacerbated by, or caused by,
excessive or unregulated cytokine production.

35 As used herein, the cytokine referred to in the phrase "inhibition of a cytokine, for

use in the treatment of a HIV-infected human" is a cytokine which is implicated in (a) the
initiation and/or maintenance of T cell activation and/or activated T cell-mediated HIV

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gene expression and/or replication and/or (b) any cytokine-mediated disease associated

problem such as cachexia or muscle degeneration.

As TNF-3(also known as lymphotoxin) has close structural homology with TNF -

a (also known as cachectin) and since each induces similar biologic responses and binds
5 to the same cellular receptor, both TNF-a and TNF-6 are inhibited by the compounds of

the present invention and thus are herein referred to collectively as "TNF" unless

specifically delineated otherwise.

In order to use a compound of formula (I) or a pharmaceutically acceptable salt

thereof in therapy, it will normally be formulated into a pharmaceutical composition in
10 accordance with standard pharmaceutical practice. This invention, therefore, also relates

to a pharmaceutical comjxjsition comprising an effective, non-toxic amount of a

compound of formula (I) and a pharmaceutically acceptable carrier or diluent.

Compounds of formula (I), pharmaceutically acceptable salts thereof and

pharmaceutical compositions incorporating such may conveniently be administered by
1 5 any of the routes conventionally used for drug administration, for instance, orally,

topically, parenterally or by inhalation. The compounds of formula (I) may be

administered in conventional dosage forms prepared by combining a compound of

formula (I) with standard pharmaceutical carriers according to conventional procedures.

The compounds of formula (I) may also be administered in conventional dosages in
20 combination with a known, second therapeutically active compound. These procedures

may involve mixing, granulating and compressing or dissolving the ingredients as

appropriate to the desired preparation. It will be appreciated that the form and character

of the pharmaceutically acceptable character or diluent is dictated by the amount of active

ingredient with which it is to be combined, the route of administration and other well-
25 known variables. The carrier(s) must be "acceptable" in the sense of being compatible

with the other ingredients of the formulation and not deleterious to the recipient thereof.

The pharmaceutical carrier employed may be, for example, either a solid or liquid.

Exemplary of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin,

acacia, magnesium stearate, stearic acid and the like. Exemplary of liquid carriers are
30 syrup, peanut oil, olive oil, water and the like. Similarly, the carrier or diluent may

include time delay material well known to the art, such as glyceryl mono-stearate or

glyceryl distearate alone or with a wax.

A wide variety of pharmaceutical forms can be employed. Thus, if a solid carrier

is used, the preparation can be tableted, placed in a hard gelatin capsule in powder or
35 pellet form or in the form of a troche or lozenge. The amount of solid carrier will vary

widely but preferably will be from about 25mg. to about Ig. When a liquid carrier is

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used, the preparation will be in the form of a syrup, emulsion, soft gelatin capsule, sterile
injectable liquid such as an ampule or nonaqueous liquid suspension.

Compounds of formula (I) may be administered topically, that is by non-systemic
administration. This includes the application of a compound of formula (I) externally to
5 the epidermis or the buccal cavity and the instillation of such a compound into the ear,
eye and nose, such that the compound does not significantly enter the blood stream. In
contrast, systemic administration refers to oral, intravenous, intraperitoneal and
intramuscular administration.

Formulations suitable for topical administration include liquid or semi-liquid

10 preparations suitable for penetration through the skin to the site of inflammation such as
liniments, lotions, creams, ointments or pastes, and drops suitable for administration to
the eye, ear or nose. The active ingredient may comprise, for topical administration, from
0.001% to 10% w/w, for instance from 1% to 2% by weight of the formulation. It may
however comprise as much as 10% w/w but preferably will comprise less than 5% w/w,

15 more preferably from 0.1% to 1% w/w of the formulation.

Lotions according to the present invention include those suitable for application to
the skin or eye. An eye lotion may comprise a sterile aqueous solution optionally
containing a bactericide and may be prepared by methods similar to those for the
preparation of drops. Lotions or liniments for application to the skin may also include an

20 agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a
moisturizer such as glycerol or an oil such as castor oil or arachis oil.

Creams, ointments or pastes according to the present invention are semi-solid
formulations of the active ingredient for external application. They may be made by
mixing the active ingredient in finely-divided or powdered form, alone or in solution or

25 suspension in an aqueous or non-aqueous fluid, witii the aid of suitable machinery, with a
greasy or non-greasy base. The base may comprise hydrocarbons such as hard, soft or
liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin
such £is almond, com, arachis, castor or olive oil; wool fat or its derivatives or a fatty acid
such as steric or oleic acid together with an alcohol such as propylene glycol or a

30 macrogel. The formulation may incorporate any suitable surface active agent such as an
anionic, cationic or non-ionic surfactant such as a sorbitan ester or a polyoxyethylene
derivative thereof. Suspending agents such as natural gums, cellulose derivatives or
inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may
also be included.

3 5 Drops according to the present invention may comprise sterile aqueous or oily

solutions or suspensions and may be prepared by dissolving the active ingredient in a
suitable aqueous solution of a bactericidal and/or fungicidal agent and/or any other

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suitable preservative, and preferably including a surface active agent. The resulting
solution may then be clarified by filtration, transferred to a suitable container which is
then sealed and sterilized by autoclaving or maintaining at 98-100" C. for half an hour.
Alternatively, the solution may be sterilized by filtration and transferred to the container
5 by an aseptic technique. Examples of bactericidal and fungicidal agents suitable for
inclusion in the drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium
chloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for the preparation
of an oily solution include glycerol, diluted alcohol and propylene glycol.

Compounds of formula (I) may be administered parenterally, that is by

10 intravenous, intramuscular, subcutaneous intranasal, intrarectal, intravaginal or

intraperitoneal administration. The subcutaneous and intramuscular forms of parenteral
administration are generally preferred. Appropriate dosage forms for such administration
may be prepared by conventional techniques. Compounds of formula (I) may also be
administered by inhalation, that is by intranasal and oral inhalation administration.

1 5 Appropriate dosage forms for such administration, such as an aerosol formulation or a
metered dose inhaler, may be prepared by conventional techniques.

For all methods of use disclosed herein for the compounds of formula (I), the
daily oral dosage regimen will preferably be from about 0.1 to about 80 mg/kg of total
body weight, preferably from about 0.2 to 30 mg/kg, more preferably from about 0.5 mg

20 to 15mg. The daily parenteral dosage regimen about 0. 1 to about 80 mg/kg of total body
weight, preferably from about 0.2 to about 30 mg/kg, and more preferably from about 0.5
mg to 15mg/kg. The daily topical dosage regimen will preferably be from 0.1 mg to 150
mg, administered one to four, preferably two or three times daily. The daily inhalation
dosage regimen will preferably be from about 0.01 mg/kg to about 1 mg/kg per day. It

2 5 will also be recognized by one of skill in the art that the optimal quantity and spacing of

individual dosages of a compound of formula (I) or a pharmaceutically acceptable salt
thereof will be determined by the nature and extent of the condition being treated, the
form, route and site of administration, and the particular patient being treated, and that
such optimums can be determined by conventional techniques. It will also be appreciated
30 by one of skill in the art that the optimal course of treatment, i.e., the number of doses of
a compound of formula (I) or a phannaceutically acceptable salt thereof given per day for
a defined number of days, can be ascertained by those skilled in the art using
conventional course of treatment determination tests.

The invention will now be described by reference to the following examples

3 5 which are merely illustrative and are not to be construed as a limitation of the scope of

the present invention.

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BIOLOGICAL EXAMPLES

The cytokine-inhibiting effects of compounds of the present invention are
determined by the following in vitro assays:
Interleukin 1 (IL-1)

5 Human peripheral blood monocytes are isolated and purified from either fresh

blood preparations from volunteer donors, or from blood bank buffy coats, according to
the procedure of Colotta et al, J Immunol, 132, 936 (1984). These monocytes (IxlO^)
are plated in 24-well plates at a concentration of 1-2 million/ml per well. The cells are
allowed to adhere for 2 hours, after which time non-adherent cells are removed by gentle

10 washing. Test compounds are then added to the cells for Ih before the addition of

lipopolysaccharide (50 ng/ml), and the cultures are incubated at 37° C for an additional
24h. At the end of this period, culture supematants are removed and clarified of cells and
all debris. Culture supematants are then immediately assayed for IL-1 biological activity,
either by the method of Simon et al, J. Immunol. Methods, 84, 85, (1985) (based on

15 ability of IL-1 to stimulate a Interleukin 2 producing cell line (EL-4) to secrete IL-2, in
concert with A23187 ionophore) or the method of Lee et al, J. ImmunoTherapy, 6 (1), 1-
12 (1990) (ELISA assay).

Tumor Necrosis Factor (TNF)

20 Human peripheral blood monocytes are isolated and purified from either blood

bank buffy coats or plateletpheresis residues, according to the procedure of Colotta, R. et
al., J Immunol, 132(2), 936 (1984). The monocytes are plated at a density of 1x10^
cells/ml medium/well in 24-well multi-dishes. The cells are allowed to adhere for 1 hour
after which time the supernatant is aspirated and fresh medium (1ml, RPMI-1640,

25 Whitaker Biomedical Products, Whitaker, CA) containing 1% fetal calf serum plus

penicillin and streptomycin (10 units/ml) added. The cells are incubated for 45 minutes
in the presence or absence of a test compound at InM-lOmM dose ranges (compounds
were solubilized in dimethyl sulfoxide/ethanol, such that the final solvent concentration
in the culture medium is 0.5% dimethyl sulfoxide/0.5% ethanol). Bacterial lipopoly-

30 saccharide (£. coli 055:B5 [LPS] from Sigma Chemicals Co.) is then added (100 ng/ml in
10 ml phosphate buffered saline) and cultures incubated for 16-18 hours at 37° C in a 5%
CO2 incubator. At the end of the incubation period, culture supematants are removed

from the cells, centrifuged at 3000 rpm to remove cell debris. The supematant is then
assayed for TNF activity using either a radio-immuno or an ELISA assay, as described in
35 WO 92/10190 and by Becker et al, J Immunol, 1991, 147, 4307.

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Interleukin 8 (IL-8)

Primary human umbilical cord endothelial cells (HUVEC) (Cell Systems, Kirland,
Wa) are maintained in culture medium supplemented with 15% fetal bovine serum and
1% CS-HBGF consisting of aFGF and heparin. The cells are then diluted 20-fold before
5 being plated (250^.1) into gelating coated 96-well plates. Prior to use, culture medium is
replaced with fresh medium (200^.1). Buffer or test compound (25^1, at concentrations
between 1 and 10M.M) is then added to each well in quadruplicate wells and the plates
incubated for 6h in a humidified incubator at 37° C in an atmosphere of 5% CQ2. At the
end of the incubation period, supernatant is removed and assayed for IL-8 concentration
10 using an IL-8 ELISA kit obtained from R&D Systems (Minneapolis, MN). All data is

presented as mean value (ng/ml) of multiple samples based on the standard curve. ICso's
where appropriate are generated by non-linear regression analysis.

Cytokine Specific Binding Protein Assay

15 A radiocompetitive binding assay was developed to provide a highly reproducible

primary screen for structure-activity studies. This assay provides many advantages over
the conventional bioassays which utilize freshly isolated human monocytes as a source of
cytokines and ELISA assays to quantify them. Besides being a much more facile assay,
the binding assay has been extensively validated to highly correlate with the results of die

20 bioassay. A specific and reproducible CSAID binding assay was developed using soluble
cystosolic fraction from THP.l cells and a radiolabeled compound. For instance, a
suitable radiolabeled compound of the CSAID class is 4-(Fluorophenyl)-2-(4-
hydroxyphenyl-3,5-t2)-5-(4-pyridyl)imidazole. In brief, the THP.l cytosol was routinely
prepared from cell lysate obtained by nitrogen cavitation followed by a 10 K x g low

2 5 speed and a 1(X) K x £ high speed centrifugation, die supernatant of which was designated

as the cytosolic fraction. THP.l cytosol was incubated with appropriately diluted
radioligand at room temperature for a pre-determined time to allow the binding to achieve
equilibrium. The sample was added to a G-10 column and eluted with 20 mm TRN,
50mMb - mercaptoethanol, NaN3 The fraction encompassing the void volume was
30 collected and the radioactivity was assessed by liquid scintillation counting. This was

determined to reflect bound radioligand since the radioactive signal was abrogated by the
presence of excess cold ligand in the incubation mixture or when there was no cytosolic
fraction present. Compounds of Formula (I) at various doses are added to the binding
assay to achieve inhibition of binding of the radiolabel. IC50S as well as Ki values are

3 5 determined by regression analysis and scatchard plot analysis respectively. There is

generally excellent correlation between the IC50 of compounds tested in both die binding
assay and the bioassay and can be used interchangeably in many cases.

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Compounds of Formula (I), as illustrated by Example 2, herein was shown to have
activity in the CSBP assay.

08/250,975 published as WO 95/07922 on 23 March 1995 whose disclosures are
incorporated by reference herein in its entirety describes the above noted method for
screening drugs to identify compounds which interact with and bind to the CSBP.
However, for purposes herein the binding protein may be in isolated form in solution, or

10 in immobilized form, or may be genetically engineered to be expressed on the surface of
recombinant host cells such as in phage display system or as fusion proteins.
Alternatively, whole cells or cytosolic fractions comprising the CSBP may be employed
in the screening protocol. Regardless of the form of the binding protein, a plurality of
compounds are contacted with the binding protein under conditions sufficient to form a

15 compound/ binding protein complex and compound capable of forming, enhancing or
interfering with said complexes are detected.

MATERIALS :

20 Incubation buffer: 20 mM Tris, 1 mM MgCl2, 20 mM Hepes, 0.02% NaN3, store at 4''C.
Elution buffer: 20 mM Tris, 50 mM 2-mercaptoethanol, NaN3, store at 4"C.

G-10 Sephadex: add 100 g Sephadex G-10 (Pharmacia, Uppsala, Sweden) to 400 mL dd
H2O and allow to swell at room temperature for 2 hours. Decant fines and wash 3 times.
Add NaN3 and qs with dd H2O to 500 mLs and store at 4°C.

25 Assemble Columns: Straw column, filter frit and tip (Kontes, SP 420160-000, 420162-
002). Lowsorb tubes (Nunc) used in binding reaction. THP.l cytosol spun at 15(X)0 rpm
for 5 min to clarify. THP.l cytosol prepared by hypnotic treatment of cells and lysis by
decompression in nitrogen. Nuclei and membrane fiagments removed by differential
centrifiagation (10,000 g for 1 hour and 100,000 g for 1 hour).

30 Compounds: Non-radioactive Compound I with corresponding EtOH control (dilutions
made in incubation buffer) and ^H-Compound I (dilutions in incubation buffer)

5

Patent Application USSN 08/123,175 Lee et al., filed 17 September 1993 and

More specifically, the CSAID Binding Assay is performed as follows:

METHOD:

35

A.

Column Preparation

1 . Begin 30 min before anticipated elution of reaction mixture.

2. Add 3 mL of G-10 slurry to column for bed vol of 1.5 ml.

3. Rinse with 7 mL elution buffer (fill to top of column)

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4. Cut columns down to size.

B. Sample Incubation

1. 15 min incubation at 4''C.

2. Binding reaction mixture; 100 |iL cytosol, 10 uL cold Compound I
5 or EtOH control, 10 |iL ^H-Compound I (molar concentration

depends on nature of study).

3. "Free" control = 100 ^iL incubation buffer in lieu of cytosol
preparation.

C. Sample Elution

10 1. Eluteat4°C.

2. Add total reaction volume to G-10 column.

3. Add 400 \iL elution buffer to column and discard eluate.

4. Add 500 |iL elution buffer to column, collecting eluted volume in
20 ml scintillation vial.

15 5. Add 15 mL Ready Safe scintillation fluid.

6. Vonex and count in liquid scintillation counter for 5 minutes.
Include a "total input counts control" (10 |iL of labeled ligand).

D. Data Analysis

1. Plot DPMS as ouptut in graphic form and analyze by regression analysis
20 and "Lundon ligand binding" software for the determination of IC 50 and

Kd/Ki respectively.

2. Rank order the IC50s of the tested compounds in the CSAIDbioassay and
compare to that generated by the CSAID binding assay and establish a
correlation curve.

2 5 The binding assay was further validated by the following criteria: THP. 1 cytosol
demonstrated samrable and specific binding of the radiolabeled compound.

Preparation of 4- (Fluorophenyl)-2-(4-hydroxyphenyl-3,5-t2)-5-(4-pyridyl)imidazole,
(Compound I). A 2.9 mg (0.0059 mmol) portion of 2-(3,5-Dibromo-4-hydroxyphenyl)-4-
30 (4-fluorophenyl)-5-(4-pyridyl)imidazole, Compound I(p), was dissolved in 0.95 mL of
dry DMF and 0.05 mL of triethylamine in a 2.4 mL round bottom flask equipped with a
small magnetic stirring bar. A 1.7 mg portion of 5% Pd/C (Engelhard lot 28845) was
added, and the flask was attached to the stainless steel tritium manifold. The mixmre was
degassed tiirough four freeze-pump-thaw cycles, then tritium gas (5.3 Ci, 0.091 mmol)
35 was introduced. The reaction mixture was allowed to warm to room temperature and was
stirred vigorously for 20h. The mixture was frozen in liquid nitrogen, the remaining
tritium gas (2.4 Ci) was removed, and the flask was removed from the manifold. The

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reaction mixture was transferred, using 3x1 mL of methanol as rinsings, into a 10 mL
round bottom flask, and the solvents were removed by static vacuum transfer. A 1.5 mL
portion of methanol was added to the residue, then removed by static vacuum transfer.
The latter process was repeated. Finally, the residue was suspended in 1.5 mL of ethanol
5 and filtered through a syringe-tip Milliporc filter (0.45 micron), along with 3 x ca. 1 mL
ethanol rinsings. The total filtrate volume was determined to be 3.9 mL, and the total
radioactivity, 94.2 mCi. Solution was determined to be 3.9 mL, and the total
radioactivity, 94.2 mCi. HPLC analysis of filtrate (Partisil 5 ODS-3, 4.6 mm LD. x 25
cm, 1 mL/min of 70:30:01 water/acetonitrile/trifluoroacetic acid, Radiomatic Ro-One
10 Beta radio detector with 3 mL/min of Ecoscint-H cocktail through a 0.75 mL cell)

showed the presence of Compound I (R^ = 60 min. ca. 37% of total radioactivity), and a
discrete intermediate presumed to be the monobromo derivative Compound la (R^ = 1 1.8
min, ca. 9%).

The filtrate solution was evaporated to near dryness with a stream of nitrogen, and
15 the residue was dissolved in about 1.2 mL of the HPLC mobile phase. The solution was
separated by HPLC as shown below, and the peaks corresponding to Compounds I and la
are collected separately.

HPLC Method

Column Altex Ultrasphere

10 mm I.D. X 25 cm
Mobile Phase 70:30:0.1

water/acetonitrile/trifluoroacetic acid
Flow Rate 5 mL/min

UV detection 210nm
Injection Volumes 0.05 - 0.4 m:

Retention Times 7.8 min Compound I

24 min Compound la

20 The pooled Compound I fractions totaled 32 mL in volume and the radioactive

concentration was 1.52 mCi/mL (total 48.6 m Ci). The pooled SB Compound la [3h]
fractions (totaling 10.1 mCi) were evaporated to dryness and tiie residue was transferred
quantitatively into a glass vial using 3.8 mL of absolute ethanol for further analysis.

An 8 mL (12.2 mCi) portion of Compound I was evaporated to dryness in vacuo

25 at <35''C, then redissolved in 0.5 mL of mobile phase. The whole volume was injected
into the HPLC system described above, and the appropriate peak was collected.
Evaporation of the collected eluate in vacuo at <35''C and transfer of the yellow residue
into a vial with absolute ethanol provided a solution (3.8 mL, 2.44 mCi/mL) of
Compound L The portion of this solution used for NMR analyses was first evaporated to

30 dryness using stream of nitrogen then taken up in CD3OD.

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Analysis of 4-(4-Fluorophenyl)-2-(4-hydroxyphenyl-3,5-t2)-5-(4-
pyridyl)imidazole. Compound I.

Radiochemical Purity by HPLC
Method
Column

Mobile Phase

Flow Rate
Mass detection
Radioactivity detection

Scintillator

Flow rate

Cell volume
Retention time

Ultrasphere Octyl, 5mm, 4.6 mm
I.D. X 25 cm, Beckman
350:150:0.5(v/v/v)
water/acetonitrile/trifluoroacetic acid
1.0 mlVmin
UVat210nm

Ramona-D radioactivity flow detector
Tru-Count (Tru-Lab Supply Co.)
5.0 mlVmin
0.75 mL
7.7 min
98.7

Radioactive Concentration hv Scin tillation Conntin{ T
Method

Scintillator Ready Safe (Beckman Instruments, Inc.)

Instrument TM Analytic model 6881

Efficiency Automated DPM calculation from quench curve

Result 2.44 mCi/mL

Specific Activity hv Mass Snectrnmfttrv

Method
Result

3 h NMR 9

Method

Instrument
Experiment

CI-MS, NH3 reagent gas

20.0 Ci/mmol

3h Distribution:

Unlabeled 44%
Single Label 43%
Double Label 13%

Brunker AM 400
Proton decoupled 3h NMR
Proton non-decoupled NMR
Proton non-decoupled NMR
Solvent Peak of methanol 6 3.3
Methanol-d4

Tritium is incorporated exclusively on the carbon
atoms ortho to aromatic hydroxyl group
Analytical Summary
Assay Result
Radiochemical purity determined by HPLC 98.7%
Radioactivity concentration determined by scintillation 2.44 mCi/mL
counting

Peak Referencing
Solvent
Result

Specific activity determined by mass spectrometry
3h NMR

20.0 Ci/mmol

agrees with the proposed

structure

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The above description fully discloses the invention including preferred
embodiments thereof. Modifications and improvements of the embodiments specifically
disclosed herein are within the scope of the following claims. Without further elaboration,
5 it is believed that one skilled in the art can, using the preceding description, utilize the
present invention to its fullest extent. Therefore the Examples herein are to be construed
as merely illustrative and not a limitation of the scope of the present invention in any
way. The embodiments of the invention in which an exclusive property or privilege is
claimed are defined as follows.

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What is claimed is:

1. A compound of the formula:

wherein

one of Ri and R2 is selected from is 4-pyridyl, 4-pyrimidinyl, 4-quinolyl, 4-

10 isoquinolinyl, 4-quinazolinyl, which is optionally substituted with one or two

substituents each of which is independently selected from C1.4 alkyl, halo, C1-4
alkoxy, C1-4 alkylthio, CH2OR8, NH2, mono- or di-Ci-6-alkylamino or N-
heterocyclyl ring which ring has from 5 to 7 members and optionally contains an
additional heteroatom selected from oxygen, sulfur or NR22; and

15 the other of Ri and R2 is selected from an optionally substituted aryl or optionally
substituted heteroaryl group, provided that both Rj and R2 are not the same
heteroaryl group; wherein when one Ri and R2 is an optionally substituted aryl ring,
the ling is substituted by one or two substituents, each of which is independentiy
selected, and which, for a 4-phenyl, 4-naphth-l-yl or 5-naphth-2-yl substituent, is

20 halo, nitro, cyano, -C(Z)NR7Ri7, -C(Z)OR23. -(CRl0R20)nCOR36, -SR5, -S(0)R5,

-OR36, halo-substituted C1-4 alkyl, C1-4 alkyl, -ZC(Z)R36, -NRioC(Z)R23, or
-(CRioR20)n NR10R2O and which, for other positions of substitution, is halo,
-(CRioR20)n nitro, -(CRioR20)ncyano, -(CRioR20)nC(Z)NRi6R26.
-(CRioR20)nC(ZX)Rl8. -(CRl0R20)n COR25. -(CRl0R20)n-S(O)mR8,

25 -(CRioR20)nOH, -(CRioR20)nOR25. halo-substituted-Ci-4 alkyl, -C1-4 alkyl,

-(CRioR20)nNRloC(Z)R25, -(CRl0R20)nNHS(O)mR6.
-(CRi0R20)nNHS(O)niNR7Ri7, -(CRioR20)nNR6S(0)niR6,
-(CRioR20)nNR6S(0)m'NR7Ri7; -(CRioR20)nZC(Z)Ri8 or
-(CRioR20)nNR7Ri7; and when one of Ri and R2 is an optionally substituted

30 heteroaryl group, the substituent groups include one or two substituents each of which

is independentiy selected from C1-4 alkyl, halo, C1-4 alkoxy, C1-4 alkyltiiio,
NRioR20> or an N-heterocyclyl ring which ring has from 5 to 7 members and
optionally contains an additional heteroatom selected from oxygen, sulfur or NR12;

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n is 0 or an integer of 1 or 2;
n' is 0 or an integer having a value of 1 to 10;
m is 0 or an integer of 1 or 2;
m' is an integer of 1 or 2;
5 m" is an integer having a value of 1 to 10;
R3 isQ-(Yi)t;

Q is an aryl or heteroaryl group;

t is an integer having a value of 1 to 3;

R4 is hydrogen, Ci-iQalkyl, halo-substituted Ci-io alkyl, C2-10 alkenyl, C2-10 alkynyl,

10 C3-7 cycloalkyl, Cs.ycycloalkylCi-io alkyl, C5-7 cycloalkenyl, Cs-ycycloalkenyl-

Ci-io alkyl, aiyl, arylCi-io alkyl, heteroaryl, heteroaryl-Ci-iQ-alkyl, heterocyclyl,
heterocyclylCi- 10 alkyl, -(CRioR20)n' OR12, (CRioR20)nORi3,
(CRioR20)nS(0)mRl8, (CRl0R20)nNHS(O)2Rl8, (CRl0R20)nNRi6R26.
(CRioR20)nN02, (CRioR20)nCN, (CRioR20)n'S02Rl8,

15 (CRioR20)nS(0)ni'NRi6R26. (CRl0R20)nC(Z)Ri3, (CRioR20)nOC(Z)Ri3,

(CRioR20)nC(Z)ORi3, (CRioR20)nC(Z)NRi6R26, (CRlGR20)nC(Z)NRi3OR9,
(CRioR20)nNRioC(Z)Rn, (CRloR20)nNRioC(Z)NRi6R26.
(CRioR20)nN(OR6)C(Z)NRi6R26. (CRl0R20)nN(OR6)C(Z)Ri3,
(CRioR20)nC(=NOR6)Rl3, (CRl0R20)nNR ioC(=NRi9)NRi6R26.

20 (CRioR20)nOC(Z)NRi6R26, (CRloR20)nNRioC(Z)NRi6R26,

(CRioR20)nNRioC(Z)ORio, 5-(Ri8)-l,2,4-oxadizaol-3-yl or 4-(Ri2)-5-(Ri8Ri9)-
4,5-dihydro-l,2,4-oxadiazol-3-yl; wherein the aryl, arylalkyl, heteroaryl, heteroaryl
alkyl, cyclcoalkyl, cycloalkyl alkyl, heterocyclic and heterocyclic alkyl groups may
be optionally substituted;

25 R6 is Ci-10 alkyl, C3-7 cycloalkyl, aryl, arylCi-iQalkyl, heterocyclyl, heterocyclyl
Ci-ioalkyl, heterocyclyl-Ci-iQalkyl, heteroaryl or heteroarylCi-ioalkyl;
R5 is hydrogen, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl or NR7R17, excluding the

moieties -SR5 being -SNR7R17 and -SOR5 being -SOH;
RI6 and R26 are independently hydrogen, or C1-4 alkyl or R16 and R26 together with

30 the nitrogen to which they are attached form a heterocyclic or heteroaryl ring of 5-7

members optionally conatining an additional heteroatom selected from oxygen, sulfur
or NR22;

Yi is independentiy selected from hydrogen, C1-5 alkyl, halo-substituted C1-5 alkyl,
halogen, or -(CRioR20)nY2;
35 Y2 is hydrogen, halogen, -ORg, -NO2, -S(0)m'Rll, -SRg, -S(0)m'0R8, -S(0)niNR8R9,
-NR8R9, -0(CRioR20)n' NR8R9, -C(0)R8, -CO2R8,

-C02(CRioR20)m"CONR8R9, -ZC(0)R8, -CN, -C(Z)NR8R9, -NRioC(Z)R8,

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PCT/US95/06287

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-C(Z)NR80R9, -NRioC(Z)NR8R9, -NRioS(0)m'Rll, -N(OR2l)C(Z)NR8R9,
-N(OR2l)C(Z)R8, -C(=NOR2l)R8, -NRioC(=NRi5)SRii,

-NRioC(=NRi5)NR8R9, -NRioC(=CRi4R24)SRll, -NRioC(=CRi4R24)NR8R9,
-NRi0C(O)C(O)NR8R9. -NRioC(0)C(0)ORio, -C(=NRi3)NR8R9,
5 -C(=NORi3)NR8R9> -C(=NRi3)ZRi i, -OC(Z)NR8R9, -NRioS(0)mCF3,

-NRioC(Z)ORiO. 5-(Rl8)-l,2,4-oxadizaol-3-yl or 4-(Ri2)-5-(Ri8Rl9)-4,5-dihydro-
1 ,2,4-oxadiazol-3-yl;

R7 and Ri7 is each independently selected from hydrogen or C1-4 alkyl or R7 and R17

together with the nitrogen to which they are attached form a heterocyclic ring of 5 to
10 7 members which ring optionally contains an additional heteroatom selected from

oxygen, sulfur or NR22;
R8 is hydrogen, Ci-io alkyl, C3-7 cycloalkyl, heterocyclyl, heterocyclylCi-iQalkyl, aryl,

arylCi-ioalkyl, heteroaryl or heteroarylCi-iQalkyl;
R9 is hydrogen, Ci-io alkyl, C2-IO alkenyl, C2-IO alkynyl, C3-7 cycloalkyl, C5.7
15 cycloalkenyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl or Rs and R9 may together

with the nitrogen to which they are attached form a heterocyclic or heteroaryl ring of

5 to 7 members which ring optionally contains an additional heteroatom selected from

oxygen, sulfur or NR12;
RlO and R20 is each independently selected from hydrogen or C1-4 alkyl;
20 Rii is Ci-io alkyl, halo-substituted Ci-io alkyl, C2-IO alkenyl, C2-IO alkynyl, C3-7

cycloalkyl, C5-7 cycloalkenyl, heterocyclic, heterocyclic Ci-ioalkyl, aryl, aryl

Ci-ioalkyl, heteroaryl or heteroaryl Ci-iQalkyl;
R12 is hydrogen, -C(Z)Ri3, optionally substituted C1-4 alkyl, optionally substituted aryl,

optionally substituted aryl-Ci-4 alkyl, or S(0)2Rl8;
25 Ri3 is hydrogen, Ci-io alkyl, C3-7 cycloalkyl, heterocyclyl, heterocyclylCi-io alkyl

aryl, arylCi-iQalkyl, heteroaryl or heteroarylCi-iQalkyl;
Rl4 and R24 is each independently selected from hydrogen, alkyl, nitro or cyano;
Rl5 is hydrogen, cyano, C1-4 alkyl, C3-7 cycloalkyl or aryl;

RI6 and R26 is each independently selected from hydrogen or optionally substituted
30 C1.4 alkyl, optionally substituted aryl or optionally substituted aryl-Ci-4 alkyl, or

together with the nitrogen which they are attached form a heterocyclic ring of 5 to 7
members which ring optionally contains an additional heteroatom selected from
oxygen, sulfur or NR12;
RI8 is Ci-lO alkyl, C3-7 cycloalkyl, aryl, arylCi-ioalkyl, heterocyclyl, heterocyclyl-
35 Ci-ioalkyl, heteroaryl or heteroarylCi-iQalkyl;

Rl9 is hydrogen, cyano, Ci^ alkyl, C3-7 cycloalkyl or aryl;

wo 95/31451

PCT/US95/06287

-36-

R21 is hydrogen, a pharmaceutically acceptable cation, Ci-io alkyl, C3.7 cycloalkyl,
aryl, arylCi.4 alkyl, heteroaryl, heteroarylalkyl, heterocyclyl, aroyl, or Ci-io
alkanoyl;

R22 is RlO or C(Z)-Ci-4 alkyl;
5 R23 is C1.4 alkyl, halo-substituted-C I-4 alkyl, or C3-7 cycloalkyl;

R25 is Ci-io alkyl, halo-substituted Ci-io alkyl, C2-IO alkenyl, C2-IO alkynyl, C3.7
cycloalkyl, C5-7 cycloalkenyl, aiyl, arylCi-io alkyl, heteroaryl, heteroarylCi-io
alkyl, heterocyclyl, hetcrocyclylCi-io alkyl, (CRioR20)nOR8,
(CRioR20)nS(0)mRl8. (CRl0R20)nNHS(O)2Rl8, (CRioR20)nNR7Rl7; wherein
10 the aryl, arylalkyl, heteroaryl, heteroaryl alkyl may be optionally substituted;

R36 is hydrogen or R23;

or a pharmaceutically acceptable salt thereof.

2. The compound according to Claim 1 wherein Rj is an optionally substituted 4-
1 5 pyridyl or 4-pyTimidinyl.

3. The compound according to Claim 2 wherein the optional substituent is C1-4
alkyl, amino, or mono substituted C1.6 alkyl amino.

20 4. The compound according to Claim 3 wherein R] is 4-pyrimidinyl.

5. The compound according to Claim 1 wherein R2 is an optionally substituted
phenyl.

25 6. The compound according to Claim 5 wherein one or more optional substituents
are independently selected ftom halogen or methoxy.

7. The compound according to any of Claims 1 to 6 wherein R4 is hydrogen or
NR16R26-

30

8. The compound according to Claim 7 wherein R16 and R26 are hydrogen,
optionally substituted C1-4 alkyl, or R16 and R26 together with the nitrogen to which
they are attached form a 5 to 7 member heterocyclic ring, optionally containing an
additional heteroatom selected from O/N/S.

35

9. A pharmaceutical composition comprising a pharmaceutically acceptable carrier
or diluent and a compound according to Claim 1 .

wo 95/31451

PCT/US95/06287

-37-

10. The compound according to any of Claims 1 to 9 which is 4-(2-Amino-4-
pyrimidinyl)-3-(4-fluorophenyl)- 1 -phenylpyrazole.

5 11. A method of treating a cytokine mediated disease in an animal in need thereof
which method comprises administering to said animal an effective cytokine mediating
amount of a compound according to Claim 1 .

12. The method according to Claim 1 1 wherein the cytokine mediated disease is
10 asthma, adult respiratory distress syndrome, bone reasorption diseases, arthritic joint

conditions, and other inflammatory diseases.

13. The method according to Claim 12 wherein the mediation of the disease state is
by the cytokine Interleukin-1 (IL-1), cytokine Interleukin-8 (IL-8), or the cytokine Tumor

1 5 Necrosis Factor (TNF).

14. The method according to any of Claims 1 1 to 13 wherein the compound is 4-(2-
Amino-4-pyrimidinyl)-3-(4-fluorophenyl)- 1 -phenylpyrazole.

20

15. A method of treating inflammation in a mammal in need thereof which comprises
administering to said mammal an effective amount of a compound according to Claim 1.

INTERNATIONAL SEARCH REPORT

PCT/US95/06287

A. CLASSIFICATION OF SUBJECT MATTER

IPC(6) :C07D 401/04 ; A61K 31/44

US CL : 546/279 ; 514/341

According to International Patent Classification (IPC) or to both national classification and IPC

B. HELDS SEARCHED

Minimum documenution searched (classification system followed by classification symbols)

U.S. : 546/279 ; 514/341

Documentation searched other than mmimum documentaUon to the extent that such documents are included in the fields searched

NONE

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)
CAS ONLINE

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category*

Citation of document, with indication, where appropriate, of the relevant passages

Relevant to claim No.

A

J. HETEROCYCLIC CHEM., Volume 24, May-June (1987) ,
Lentz et al. "The Preparation of N-Carboalkoxyprazoles and
N-Phenylpyrazoles from C( )-Dianions of Carboxyhydrazones
and Phenylhydrazones", pages 555-558,

1-9, 11-15 (in
part)

A

US, A, 5,051,518 (MURRAY et al.) 24 September 1991.

1-9, 11-15 (in
part)

□

Further documents are listed in the continuation of Box C. | j Sec patent family annex.

Special cueg orie* of cited doctimenti:

dociimentdefiiiinf llie (eoenl itate of Ifae tit whicfa ■ not ooMidefed
to be put of panicular fclevuce

earlier document publkhed oa or after ifae inleraaliooal iilint

document whicfa may dirow doubla on priori^ elaim(i) or whiA ■
ciua to catablidi tlic publicalion dale of anolfaer citatioa or odier
•pecial reaaoo (a* ipecified)

document referring to an oral diacloaure. use, exfajbitioa or odMsr

later document publidied after Ifae mlenalaoaal filinc date or priority
date and not in conflict with Ifae applicaiioa but cited to undemand the
p(BC%>le or Ifaeofy underlyinc d>e invention

document of particular lelevaaoe: Ifae claimed invenlian cannot be
coosidend novel or cannot be cooaidered to mvolve an invcniive aiep
when Ifae document •■ laken alone

document of paitinilar lelevanoe; Ifae claimed inveniiaa cannot be
comidered to mvolve an invenlive ttcp when Ifae document it
combined wilfa one or more odier nicfa documenti. mcfa combinatioa
beinc obviout to a penoo akilkd in the art

document publiihed prior to the international filinf date but later t

the priority date claimed

donimrnt mfmbrr of Ifae

patent iamily

Date of the actual completion of the international search
16 AUGUST 1995

Date of mailing of the international search report

ISSEP 1995

Name and mailing address of the ISA/US
Commissioner of Patents and Trademarks
Box PCX

Washington. D.C. 20231
Facsimile No. (703) 305-3230

Authoruetf officer / - ^

JANE T. FAN
Telephone No. (703) 308-1235

Form PCT/ISA/210 (second sheet)(July 1992)*

INTERNATIONAL SEARCH REPORT

Intenuaional application No.
PCT/US95/06287

Box I Observations where certain claims were found unsearchable (Continuation of item 1 of first sheet)

This international report has not been established in respect of certain claims under Aitiole 17(2Xa) for the following reasons:

1. I j Claims Nos.:

because they relate to subject matter not required to be searched by this Authority, namely:

2. ^1 Claims Nos.:

because they relate to parts of the international application that do not comply with the prescribed requirements to such
an extent that no meaningful international search can be carried out, specifically:

3. Claims Nos.:

because they are dq>endent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a).

Box II Observations where unity of invention is lacking (Continuation of item 2 of first sheet)

This International Searching Authority found multiple inventions in this international application, as follows:
Please See Extra Sheet.

I I •'^ required additional search fees were timely paid by the applicant, this intcmaUonal search report covers all searchable

claims.

2. Q As all searchable claims could be searched without effort justifying an additional fee, this Authority did not invite payment
of any additional fee.

3 . As only some of the required additional search fees were timely paid by the applicant, this international search rt:port covers
only those claims for which fees were paid, specifically claims Nos.:

\_x\ No required additional search fees were timely paid by the applicant. Consequently, this international search report is
restricted to the invention first mentioned in the claims; it is covered by claims Nos.:
1-9,1 l-15(in part) wherein one of R1,R2 is pyridyl, no additional hetero anywhere.

Remark on Protest | | The additional search fees were accompanied by the applicant's protest.

I I No protest accompanied the payment of additional search fees.

Form PCT/ISA/210 (continuation of first sheet(l))(July 1992)*

INTERNATIONAL SEARCH REPORT

International application No.
PCTAJS95/06287

BOX n. OBSERVATIONS WHERE UNITY OF INVENTION WAS LACKING
This ISA found multiple inventions as follows:

This application contains the following inventions or groups of inventions which are not so linked as to form a single
inventive concept under PCT Rule 13.1. In order for all inventions to be examined, the appropriate additional
examination fees must be paid.

I. Claims 1-9,11-15 ( in part ) drawn to compounds, compositions, and method of using wherein one of R1,R2 is
pyridyl the other is non-hetero group, no additional hetero-group in the formula.

II. Claim 10 and claims 1-9, 11-15 ( in part ) wherein one of R1,R2 is pyrimidyl the other is non-hetero containing, no
additional hetero group containing.

III. Claims 1-9,11-15 ( in part ) wherein one of R1,R2 is quinolyl, the other is non-hetero group, no additional hetero-
group in the formula.

TV. Claims 1-9,11-15 ( in part ) wherein one of R1,R2 is boquinolinyl, the other is non-hetero group, no additional
group in the formula.

V. Claims 1-9, 11-15 ( in part ) wherein one of Rl ,R2 is qutnazoliny, the other is non-heter group, no additional hetero
group in the formula.

VI. Claims 1-9, 11-15 ( in part ) wherein there are two or more hetero groups in the formula, each hetero group and
their combination or permutation is distinct.

The inventions listed as Groups I-IV do not relate to asingleinventive concept under PCT Rule 13.1 because, under
PCT Rule 13.2, they lack the same or corresponding special technical features for the foUovtring reasons: The products
of groups I- VI are not so linked as to be obvious varients of each other and they are not interchangeable bio-isosters.
Furthermore, the core structure, the pyrazole ring is not a contribution over the prior art since compound 40 in Duncan
et al.( p. 557 ) reference is known and it pocesses other biological use.

Form PCT/ISA/210 (extra sheet)(July 1992)*